Nucleic Acid Amplification Assay and Arrangement Therefor

ABSTRACT

A nucleic acid amplification assay for quantitative and/or qualitative analysis of the presence of a specific analyte or specific analytes in a biological sample, which analytes, if present, are contained in particles ( 4 ) of the sample ( 2 ), in which assay the sample is forced in a first direction through a filter ( 6 ) that retains the particles ( 4 ). The particles ( 4 ) retained in the filter ( 6 ) are flushed, by a flow ( 8 ), in a second opposite direction through the filter ( 6 ) out of the filter ( 6 ) and the flow ( 8 ) containing the particles ( 4 ) flushed out is analyzed for the analyte or analytes. An arrangement ( 12 ) for preparing the sample ( 2 ) for analysis according to the assay of the invention and to a kit of parts for analyzing the analyte or analytes, which kit includes the arrangement ( 12 ) is also disclosed.

FIELD OF THE INVENTION

The present invention relates to nucleic acid amplification assays. Theinvention further relates to an arrangement for the assay. Morespecifically the present invention relates to nucleic acid amplificationassays with a simplified process for preparing a biological sample toenable an altogether simplified analysis of an analyte or analytes.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

During the past few decades, tremendous advances have been made in thefield of nucleic acid amplification assays. The polymerase chainreaction and other nucleic acid amplification and detection methods havemade it possible to specifically detect and quantify various biologicalentities—hereafter called analytes—in different kinds of samples.Measurement of the amount or mere presence of such analytes is ofimportance in a vast range of situations, examples of which includediagnosis and monitoring of disease in man or in animals; environmentalmonitoring; detection of biological warfare agents; forensic sciencesand detection and recognition of cells and viruses. Developments indifferent aspects of these molecular recognitiontechniques—instrumentation, label technologies, reagents andconsumables—have made it possible to detect even minute amounts ofspecific molecules of interest. Using the polymerase chain reaction(Saiki et al., Science 1985, 230: p. 1350-4) coupled with a suitabledetection method, for example, it is often possible to detect a singlenucleic acid analyte molecule of a particular base sequence in thepresence of a great excess of other sequences.

However, nucleic acid amplification assays are limited by the fact thatthe sample material itself, from which the measurements are to be made,must be purified prior to analysis. This is due to the fact that manycomponents of e.g. blood, environmental or food samples can inhibit theenzymes used in analysis; interfere with the formation of bioaffinitybonds that are essential for the assays; increase the background signalobtained in the measurement step; or otherwise compromise assayperformance. This is particularly true for DNA or RNA extraction (Lantzet al. Biotechnol. Annu. Rev. 2000: 5 p. 87-130). Common methods ofsample preparation have been reviewed recently by Radstrom et al.[Sachse K & Frey J (ed.), Methods in molecular biology, Vol 216: PCRdetection of microbial pathogens, Humana Press Inc., Totowa, USA: p.31-50]. These include biochemical methods based on, for example,extraction of nucleic acids using organic solvents, followed by ethanolprecipitation and solubilisation in an aqueous solvent; or lysis ofcells in the presence of chaotropic salts, affinity binding of thenucleic acids on a solid phase; and elution of pure nucleic acids usingan aqueous solvent. The main advantage of these biochemical extractionmethods is that the analyte is obtained in a pure form without any assayinhibitors. However, all of these methods present a real challenge toautomation, are labour intensive and require specialized, expensiveequipment together with harsh chemicals that cannot be used in, forexample, field conditions.

In addition to the possibility of assay inhibition, sample volume itselfis often a problem. Even if a sensitive assay is capable of detecting asingle analyte molecule, this is sometimes not enough. For example,according to regulations concerning some pathogenic organisms, such asbacteria belonging to the genera Salmonella or Listeria, foodstuff mustnot contain more than a single viable bacterial cell in 25 g of thefoodstuff. The amount of sample—25 g—is far too great to be analysed inone bioaffinity reaction. For this reason, the analyte must beconcentrated or/and enriched prior to analysis. Analyte concentrationcan be done by immunological or/and physical means (see R{dot over(a)}dström et al.). More often than not, physiological enrichment inselective culture media is used. Such enrichment usually takes between24 and 48 hours. In many cases, the time needed to perform the analysisis therefore very long, which results in significant storage costsbefore a product, e.g. animal feed product, can be released to market.

To simplify the sample pre-treatment protocols, several attempts havebeen made to develop methods where assay inhibitors would be removedwithout the need to extract DNA or RNA in a pure form and where theanalyte would be concentrated to a detectable level. Mainly, thesemethods are based on enrichment of the target cells from a sample, afterwhich the cells are subjected to analysis. Venkateswaran et al. (Appliedand Environmental Microbiology 1997, 63: p. 4127-4131) described the useof centrifugation and filtration to extract bacterial cells that weresubsequently subjected to analysis by PCR. Although this method alloweddetection of bacterial cells in the absence of DNA extraction, it waslimited in the sense that a centrifuge was needed, which makes themethod poorly suited for automation or for use outside a laboratory.Also, in the method described by Venkateswaran et al., target cells werecollected from the filter by resuspending them in a buffer, which mayvery well result in some cells being trapped on the filter. This meansthat the method does not allow quantitative determination of the amountof the target cells.

In summary, the use of sophisticated molecular recognition andquantitation techniques is usually only possible in specializedlaboratories, because the purification and enrichment techniques thatare required by most nucleic acid sequence detection methods are labourintensive and need specialized equipment and operator skills.

Therefore, there is a need for simple, fast and inexpensive samplepreparation methods that allow reduction of the amount of assayinhibitors in the sample together with concentration or enrichment ofanalyte molecules.

OBJECTS AND SUMMARY OF THE INVENTION

An object of the present invention is to provide a nucleic acidamplification assay with simplified process for preparing a biologicalsample to enable an altogether simplified analysis of an analyte oranalytes.

Another object of the present invention is to provide an arrangement forpreparing a biological sample according to the invention.

Still another object of the present invention is to provide a nucleicacid amplification assay kit for analysis of an analyte or analytescomprising the arrangement for preparing a biological sample accordingto the invention.

Thus the present invention provides a nucleic acid amplification assayfor quantitative and/or qualitative analysis of the presence of aspecific analyte or specific analytes in a biological sample, whichanalytes, if present, are contained in biological particles of saidsample, in which assay the sample is forced in a first direction througha filter that retains said biological particles. Characteristic for themethod is that the biological particles retained in the filter areflushed, by a flush flow, in a second opposite direction through thefilter out of the filter and the flush flow containing the biologicalparticles flushed out is analysed for the analyte or analytes.

The present invention further provides an arrangement for preparing abiological sample for quantitative and/or qualitative analysis of thepresence of a specific analyte or specific analytes, which analytes, ifpresent, are contained in biological particles of the sample wherein thearrangement comprises

a) a housing for a filter;

b) a filter within said housing for retaining the particles containingthe analyte or analytes, said filter having two sides,

-   -   i) a sample inlet side and    -   ii) a flushing flow inlet side; and

c) means for

-   -   i) leading the sample through the filter from the sample inlet        side to the flushing flow inlet side,    -   ii) leading the flush flow from its inlet side to the sample        inlet side, and    -   iii) retrieving for analysis biological particles containing the        analyte flushed from the filter.

Characteristic for the arrangement is that it comprises a filter rackthat is a multi-way valve, with connections for sample inlet, sampleretrieval, flush flow inlet and waste disposal, and optionally for washflow, and the filter rack with the filter can be turned in alternativepositions so that flow is directed from

d) the sample inlet into the filter from the sample inlet side to theflush flow inlet side and to waste or optionally for use as flush flow,

e) the flush flow inlet into the filter from the flush flow inlet sideto the sample inlet side and to sample retrieval, or

f) optionally, the flow inlet into the filter from the sample inlet sideto the flush flow inlet side and to waste or for recycling.

The present invention also provides a kit of parts, components and/orreagents for performing the assay according to the invention.Characteristic for the kit is that it comprises the arrangementaccording to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the basic principle of the invention.

FIG. 2 a and 2 b schematically show the principle of the invention usedwith pre-filtration.

FIG. 3 a, 3 b and 3 c schematically show the principle of an automaticsample pre-treatment instrument utilizing the principles of the presentinvention.

FIG. 4 shows a standard curve for the detection of Listeriamonocytogenes from milk using the present invention for samplepre-treatment and real time PCR for analyte detection.

FIG. 5 shows a standard curve for the detection of Listeriamonocytogenes from cheese using the present invention for samplepre-treatment and real time PCR for analyte detection.

FIG. 6 shows a standard curve for the detection of Listeriamonocytogenes from salted salmon using the present invention for samplepre-treatment and real time PCR for analyte detection.

FIG. 7 shows a standard curve for the detection of Bacillus subtilisfrom Luria broth using the present invention for sample pre-treatmentand real time PCR for analyte detection.

FIG. 8 shows a standard curve for the detection of Bacillus subtilisendospores from potato flour using the present invention for samplepre-treatment and real-time PCR for analyte detection.

FIG. 9 shows a picture of an agarose gel analysis of the PCRamplifications of an actin fragment from human blood leucocytes isolatedwith the method of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term nucleic acid amplification assay as used herein refers tomethods and techniques that are used to determine the presence or thepresence and quantity of an analyte molecule in a sample, which methodsand techniques include a first sample pre-treatment step and a secondamplification step and a third detection step. The amplification stepand the detection step together form an analysis step. In the samplepre-treatment step, the sample is treated so that its contents can besubjected to an analysis step. Important features of the samplepretreatment step include but are not limited to removing from thesample any interfering substances that could inhibit the subsequentanalysis step and/or adjusting the concentration of the analyte moleculeso that its concentration is suitable for analysis. In the amplificationstep, all or a part of the pre-treated sample molecules are subjected toconditions under which conditions enzymatic or chemical amplification ofa nucleic acid molecule or nucleic acid molecules occurs. The moleculeor molecules that is or are amplified can be identical to the analytemolecule or to a part of the analyte molecule, the presence or thepresence and quantity of which in the sample is being analysed, or canbe different from the analyte molecule. In the third step of the nucleicacid amplification assay, which third step is the detection step, theappearance or the appearance and quantity of the amplification productproduced in the amplification step is determined. The conditions of theamplification step are chosen so that the appearance or the appearanceand quantity of an amplification product reflects or reflect thepresence or the presence and the quantity of the analyte molecule in thesample.

In one preferred embodiment of the present invention, a nucleic acidamplification assay comprises the steps of

1) preparing a biological sample for quantitative and/or qualitativeanalysis of the presence of a specific analyte or specific analytes,which analytes, if present, are contained in biological particles of thesample, in which method the sample is forced in a first directionthrough a filter that retains said biological particles characterised inthat said biological particles retained in said filter are flushed, by aflush flow, in a second opposite direction through said filter out ofsaid filter;

2) subjecting the flush flow obtained in the previous step to conditionsunder which enzymatic or chemical amplification of one or severalnucleic acid molecules occurs, the conditions being selected so that theappearance or the appearance and the quantity of an amplified productnucleic acid or amplified nucleic acids reflects the presence or thepresence and the quantity of the analyte or analytes in the biologicalsample, said molecule or molecules being identical or different to theanalyte or analytes, the presence or the presence and the quantity ofwhich is being analysed in the biological sample; and

3) determining the appearance or the appearance and the quantity of theamplification product obtained in the previous step.

It will be appreciated by those skilled in the art that the analysisstep, consisting of an amplification step and of a detection step, canbe performed in many different ways. In one preferable embodiment of thepresent invention, the analysis step is performed by real-timepolymerase chain reaction (PCR). In real-time PCR, the presence or thepresence and quantity of a nucleic acid is determined by placing asample suspected to contain the nucleic acid in a container containingan entity capable of indicating the presence of the nucleic acid andcapable of providing a signal related to the quantity of the nucleicacid. This entity can, for example, be an intercalating dye or a probe.Next, the mixture is subjected to conditions under which the nucleicacid is amplified. The signal provided by the entity is recorded in realtime during the amplification or, alternatively, after completion of theamplification, at different temperatures, said signal being related tothe presence or the presence and quantity or the presence and quantityand quality or the presence and quality of the nucleic acid. Othersuitable ways of performing the analysis step include but are notlimited to real-time polymerase chain reaction (PCR); PCR and agarosegel electrophoresis; and PCR with homogeneous or heterogeneoushybridization detection of the PCR product using labelledoligonucleotide probes or probes made of nucleic acid analogs.Alternatively, instead of using the polymerase chain reaction foramplification, other amplification methods can be used. Other possibleamplification methods include but are not limited to reversetranscriptase PCR (RT-PCR), nucleic acid sequence based amplification(NASBA; Compton J, 1997, Nucleic acid sequence-based amplification,Nature 350:91-92), ligase chain reaction (LCR), proximity ligation assay[Gullberg M, Fredriksson S, Taussig M, Jarvius J, Gustafsdottir S,Landegren U; A sense of closeness: protein detection by proximityligation. Curr Opin Biotechnol. 2003 February; 14(1):82-6] and stranddisplacement amplification [SDA; Walker G T, Fraiser M S, Schram J L,Little M C, Nadeau J G, Malinowski D P, Strand displacementamplification—an isothermal, in vitro DNA amplification technique;Nucleic Acids Res. 1992 Apr. 11; 20(7):1691-6]. Suitable amplificationand detection methods have been discussed for example in the bookMolecular Cloning, A Laboratory Manual [Sambrook and Russell (ed.), 3rdedition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., USA].

The object of the present invention is to provide simple, fast andcost-effective sample preparation that is amenable to automation as wellas manual operation that allows purification and concentration of e.g.biological nucleic acid or protein analytes from biological,environmental or other liquid or gaseous samples. Gaseous and liquidsamples are suitable as such whereas solid samples have first to besuspended in a liquid.

A seminal finding of the present invention is that target cells can beenriched and purified from assay inhibitors by using a filter thatallows liquid or gas to be directed through the filter in two opposeddirections: the sample is first forced through the filter in such amanner that the target cells or other biological complexes of interestare retained on and/or in the filter while any contaminating orinhibitory substances pass through, after which the direction of flow isreversed and the target cells or other biological complexes arecollected and subjected to molecular analysis preferably without furtherpurification steps.

In nucleic acid assays, pre-analytical processing of samples is ofutmost importance. To analyse e.g. the protein and/or nucleic acidcontent of a sample, it is often necessary to purify and to concentratethe sample, otherwise it is impossible to determine the amount or merepresence of the specific analyte of interest in the sample. This istypically due to either presence of assay inhibitors in the sample orlow concentration of analyte or both. Most sample preparation methods,reviewed e.g. by R{dot over (a)}dström et al. (2003), are limited by thefact that they are time-consuming and labour intensive and requirespecialized equipment, operator skills and harsh chemicals.

The present invention provides simple process for sample purificationand enrichment that is amenable to automated or manual use. The generalprinciple of the process is depicted in FIGS. 1 and 2. As can be seenfrom these figures, the technique is based on separation of biologicalparticles 4, e.g. cells, containing the molecules of interest from thesample matrix using a filter 6. Optionally, the sample 2 can bepre-filtered before application to the main filter 6 in order to removelarger particles 10 (FIG. 2) that might interfere with the analysis ofthe analyte or analytes. As the biological particles 4 of interest aretrapped on and/or in the filter 6, any interfering substances, such ascompounds that could inhibit the subsequent analytical steps, passthrough. After the entire volume of sample 2 has been forced through thefilter 6, the biological particles 4 of interest can be optionallywashed by e.g. applying a suitable volume of water or other liquidthrough the filter 6 (FIG. 2). After this first step, the direction offlow 8 is reversed so that the trapped particles 4 are detached from thefilter 6. This second flush volume can be adjusted so that the particles4, now purified from inhibitors, are flushed in a volume that is smallerthan the initial sample volume, therefore resulting in effectiveenrichment or concentration of the analyte. The force and volume of thereverse flush flow 8 can be adjusted so that all or nearly all trappedanalyte containing biological particles 4 are collected. This enableseven quantitative analysis of the amount of particles 4 in the sample 2.

The flush flow 8 can be any flow, even that of the sample filtrate.Preferably it is, however, a liquid or gas flow other than the samplefiltrate in order to avoid reintroducing components of the originalsample that might interfere with the analysis of the analyte.

Flush flow in a second opposite direction through the filter as usedherein means that the direction of flow in relation to the filter planeof the sample inlet side 16 of the filter 6 is changed so that thetrapped particles are collected into the flush flow 8 filtrate afterchanging the direction of flow. As will be appreciated by those skilledin the art, this action can be accomplished in different ways. Forexample the position of the filter 6 can be changed so that thedirection of flow in relation to the filter 6 changes; or the filter canremain stationary while direction of flow is changed; or the filter 6and direction of flow can both be adjusted so that the same end result,collection of trapped biological particles 4, is achieved. The flushflow 8 can be directed toward the filter from any suitable direction, aslong as it flows out from the filter on the sample inlet side 16 and thebiological particles 4 contained in the sample 2 are first trapped inthe filter 6 and then, after changing the flow direction, collected inthe flush flow 8 filtrate.

In some applications, the sample 2 may contain interfering particles orinhibitor complexes 10 that are greater in size than the biologicalparticles 4, e.g. cells, containing the molecular species of interest.In these cases, it is possible to perform a first filtration where thebiological particles 4 of interest pass through the first filter 26while the interfering greater particles 10 are trapped in the firstfilter 26. The filtrate of the first filter 26 containing e.g. thecomplexes of interest 4 is then forced through a second filter 6 thattraps the e.g. complexes of interest while any interfering substances,such as compounds that could inhibit the subsequent analytical steps,pass through. After this, the direction of flow through the secondfilter 6 is changed and the biological particles 4 of interest arecollected and then subjected to analysis. Again, it is possible toadjust the volumes applied through the filters 26, 6 at the differentsteps to achieve optimal purity and concentration of the particles 4.

The process of the present invention can be performed manually using amanual filtration device and a manual device that allows application ofmaterial through the filter, suitably a syringe. Alternatively, theprocess of the present invention can be performed using an automateddevice that is designed to perform the physical actions necessary toforce a sample through a filter; to reverse the direction of flowthrough the filter; and to collect the biological particles. Suitably,pressure or vacuum is used to force material through a filter.

The terms biological particles as used herein refer to prokaryotic oreukaryotic cells or spores or components thereof, viral particles orcomplexes containing protein and nucleic acid, or complexes containingprotein or complexes containing nucleic acid as well as any combinationsthereof. Biological particles can e.g. be bacteria or bacterial cells,plant pollen, mitochondria, chloroplasts, cell nuclei, viruses, phages,chromosomes or ribosomes.

Retention of the biological particles in the filter can be due to theirsize and/or due to their chemical properties. Typically retention isessentially due to either the size of the particles or the chemicalproperties of the particle.

Suitably, the biological particles purified and enriched according tothe principle depicted in FIGS. 1 and 2 is analysed by a method thatallows measurement of the presence or amount of a specific molecule inthe particles. These methods include but are not limited to thefollowing: the polymerase chain reaction (PCR), reverse transcriptasepolymerase chain reaction (RT-PCR), ligase chain reaction (LCR),proximity ligation assay, oligonucleotide ligation assay (OLA), nucleicacid sequence based amplification (NASBA), strand displacementamplification (SDA). A combination of methods can also be used.

Suitably, the biological particles are flushed with a liquid or a gasthat is different from the liquid or gas originally present in thesample prior to forcing the sample through the filter. Alternatively,the biological particles are flushed with the same liquid or gas thatwas present in the sample prior to forcing the sample through thefilter.

Suitable the assay of the present invention can be used to detect aliving and/or dead cell or virus; a nucleic acid; or any combinationthereof. Typically, the method of the present invention can be used todetect one or more of the following: a bacterium, a yeast, a mould, aeukaryotic cell or organism, a cancer cell, a virus (e.g. pathogenic), anucleic acid, a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), aderivative of a nucleic acid, or a complex of protein and nucleic acid.The method can also be used to detect any combination thereof.

Suitably, the assay of the present invention is used for one or more ofthe following purposes: diagnostics, environmental monitoring, detectionof biological warfare agents, forensics, detection of micro-organisms,monitoring of industrial processes, drug discovery, development ofmedicaments, development of nutraceuticals, product quality control andgenetic analysis.

The invention also concerns a kit of parts, components and/or reagentsfor use in the assay according to the invention. Such a kit comprisesthe arrangement according to the invention and additionally other parts,components and/or reagents for performing the assay. Additional otherparts, components and/or reagents are typically tailored for a specificanalysis or a group of specific analyses. The kit can comprise a set ofessential parts, components and/or reagents, but need not compriseeverything (but the sample) needed for the analysis or analyses.Preferably it comprises all the parts, components and/or reagents nototherwise available at the typical site of carrying out the specificanalysis or analyses.

FIG. 1 shows the basic principle of the invention. Analyte particles 4are collected from a sample 2 by a filtration step. The flow through thefilter 6 is then changed in such a way that pure analyte particles 4 areflushed by the flush flow 8 from the filter 6, ready for analysis.

FIGS. 2 a and 2 b show a schematic presentation of the principle of theinvention used in a more complex way. Large sample contaminants 10 areremoved from the sample 2 with optional pre-filtration using apre-filter 26 after which the analyte particles 4 are collected from thesample 2 by filtration with a filter 6. Any small molecule contaminantsfrom the sample that may have been retained on the filter 6 are removedby an optional washing step. The flow through the filter 6 is thenreversed in such a way that pure analyte particles 4 are flushed fromthe filter by the flush flow 8 and the particles 4 are ready foranalysis.

FIGS. 3 a, 3 b and 3 c show the principle of a prototype automaticsample pre-treatment instrument utilizing the principles of the presentinvention. In FIG. 3 a the sample 2 with its analyte containingbiological particles 4 is first pumped along a flow channel 28 so thatit passes through an optional pre-filter 26, which removes large samplecontaminants. Next the sample 2 passes through a filter 6 from thesample inlet side 16 to the flush flow inlet side 18 in its housing 14mounted on a filter holder rack 32 capable of rotation. Analyteparticles 4 are thus bound on the filter 6. The filtrate of filter 6 isled to waste 38. In FIG. 3 b the filter holder rack 32 is thenoptionally rotated in such a way that wash buffer 34 can be pumpedthrough a pipe 30 through the filter 6 from the sample inlet side 16 tothe flush flow inlet side 18, removing small sample contaminantsadhering to the filter 6. The filtrate of the wash buffer is led towaste 38. In FIG. 3 b the filter holder rack 32 is finally rotated insuch a way that buffer 36 for flushing can be pumped through the filter6 in a reverse direction compared to the sample and wash buffer flow,i.e. from the flush flow inlet side 18 to the sample inlet side 16.Analyte particles 4 are thus flushed with the flush flow 8 and retrieved24 pure and ready for analysis.

FIG. 4 is a standard curve for the detection of Listeria monocytogenesfrom milk using the process of the present invention for samplepre-treatment and real time PCR for analyte detection.

FIG. 5 is a standard curve for the detection of Listeria monocytogenesfrom cheese using the process of the present invention for samplepre-treatment and real time PCR for analyte detection.

FIG. 6 is a standard curve for the detection of Listeria monocytogenesfrom salted salmon using the process of the present invention for samplepre-treatment and real time PCR for analyte detection.

FIG. 7 is a standard curve for the detection of Bacillus subtilis fromLuria broth using the process of the present invention for samplepre-treatment and real time PCR for analyte detection.

FIG. 8 is a standard curve for the detection of Bacillus subtilisendospores from potato flour using the process of the present inventionfor sample pre-treatment and real-time PCR for analyte detection.

FIG. 9 is a picture of an agarose gel analysis of the PCR amplificationsof an actin fragment from human blood leucocytes isolated with themethod of the present invention. Lanes 1 and 8 are molecular weightstandards (GeneRuler™ 100 bp DNA Ladder, Fermentas Life Sciences,Lithuania) whereas lane 7 is a positive control and lane 2 is a negativecontrol. Lanes 3 to 6 are PCR reactions with 1, 1, 10 and 10 μl ofextracted leucocytes added, respectively. The results show that the 136bp actin fragment gets amplified only in the presence of the extractedleucocytes.

Methods Bacterial strains

Listeria monocytogenes strain ATCC 7644 was used in examples 1-3.Bacillus subtilis strain 168 DE1 [Ebbole, D. J. and Zalkin, H. (1987)Cloning and Characterization of a 12-Gene Cluster from Bacillus subtilisEncoding Nine enzymes for de Novo Purine Nucleotide Synhesis. J. Biol.Chem. 262, 8274-8287] was used in examples 4 and 5.

Real-time PCR

The real-time PCR detection method used for the detection of analytes inthe examples [Nurmi, J., Wikman, T., Karp, M. and Lövgren, T. (2002)High-Performance real-Time Quantitative RT-PCR Using Lanthanide Probesand a Dual-Temperature Hybridisation Assay. Anal. Chem. 74, 3525-2532.]is based on environment sensitive terbium chelates that have greaterfluorescence intensity when they are free in solution than when attachedto single-stranded DNA. During the extension phase of PCR the5′-3′-exonucleolytic DNA polymerase digests the lanthanide probe that isspecifically hybridised to template DNA. This results in fluorescencesignal increase that is measured in a time-resolved manner with a Victor1420 Multilabel counter (Perkin Elmer Life Sciences Wallac, USA). Thesmall background fluorescence resulting from undigested lanthanideprobes is further decreased with a QSY-7-labelled quencher probe thathybridises to the terbium probe in the measurement temperature. Thethermal cycling was performed with a Peltier Thermal Cycler (MJResearch, USA).

Probe, Quencher and Primer Sequences used in PCR Reactions

Oligo- Label/ nucleotide Sequence from 5′ to 3′ end position ListeriaCGATTTCATCCGCGTGTTTCTTTTCGTA Tb/5′ probe Listeria CGCGGATGAAATCGQSY-7/3′ quencher Listeria TGCAAGTCCTAAGACGCCA None 5′primer ListeriaCACTGCATCTCCGTGGTATACTAA None 3′primer Bacillus TTGATGTGATGGCTCCTGGCCATb/5′ probe Bacillus CCATCACATCAA QSY-7/3′ quencher BacillusATGGATGTTATCAACATGAG None 5′primer Bacillus GAGTCGCCATGGACGTTC None3′primer Actine TGAAGTCTGACGTGGACATC None 5′primer ActineCTTGATCTTCATTGTGCTGGG None 3′primer

Pre-culture of Listeria Monocytogenes

Fresh Listeria monocytogenes cells were prepared as follows. An aliquotof 5 ml of brain heart infusion broth (Labema, Finland) was inoculatedwith Listeria cells and cultured overnight at 37° C. Dilutions of thesecultures were plated on nutrient agar plates (Labema) to determine theamount of Listeria cells in each batch.

EXAMPLES Example 1 Detection of Listeria Monocytogenes from Milk

Ten fold dilutions of fresh overnight Listeria monocytogenes cultureswere made to ½ Fraser broth (Labema). 20 μl of each dilution was mixedwith 1 ml of milk (2% fat content) and ½ Fraser to a total volume of 10ml. The Listeria cells were then grown for 18 h at 30° C. One ml samplesof each of these cultures were filtered through a 5 μm pore-sizepre-filter in order to remove large sample particles. Listeria cellswere then collected on a 0.45 μm pore-size filter by passing the samplethrough it. The cells on filter were washed with 10 ml of 0.9% NaCl,flow direction through the filter was reversed and the cells wereflushed with 500 μl of sterile water. 5 μl of the eluate was used in PCRreactions as template. The PCR reactions had the following conditions:1.25 U AmpliTaq Gold DNA Polymerase, 1×PCR buffer II and 5 mM MgCl2(Applied Biosystems, USA), 0.2 mM dNTPs (Amersham Biosciences, U.K.),0.3 μM Listeria primers, 0.83 μM Listeria probe and 8.3 μM Listeriaquencher in a total volume of 50 μl. The thermal cycling profile was 95°C. 10 min, 95° C. 15 s, 60° C. 1 min repeated for a total of 40 cycles.In the end of each of the last 20 cycles the temperature was brieflylowered to 35° C. for time-resolved fluorescence measuring. PCR resultswere plotted against plating results in order to obtain the standardcurve presented in FIG. 4.

Example 2 Detection of Listeria Monocytogenes from Cheese

The enrichment step was done like in example 1, except that instead ofusing milk, 1 g of blue cheese (minced thoroughly with a blender) wasmixed with the fresh Listeria cells and ½ Fraser broth. Two ml aliquotsof each of the enriched samples were filtered through a 5 μm pore-sizepre-filter in order to remove large sample particles. Listeria cellswere then collected on a 0.45 μm pore-size filter by passing the samplethrough it. The cells on filter were washed with 20 ml of 0.9% NaCl,flow direction through the filter was reversed and the cells wereflushed with 500 μL of sterile 0.9% NaCl. 5 μl of the eluate was used inPCR reactions as template. The PCR analysis was done as in example 1.PCR results were plotted against plating results in order to obtain thestandard curve presented in FIG. 5.

Example 3 Detection of Listeria Monocytogenes from Fish

The enrichment step was done like in example 1, except that instead ofusing milk, 1 g of salted salmon (minced thoroughly with a blender) wasmixed with the fresh Listeria cells and ½ Fraser broth. Two ml samplesof each of these cultures were filtered through a 5 μm pore-sizepre-filter in order to remove large sample particles. Listeria cellswere then collected on a 0.45 μm pore-size filter by passing the samplethrough it. The cells on filter were washed with 10 ml of 0.9% NaCl,flow direction through the filter was reversed and the cells wereflushed with 500 μl of sterile water. 5 μL of the eluate was used in PCRreactions as template. The PCR analysis was done as in example 1. PCRresults were plotted against plating results in order to obtain thestandard curve presented in FIG. 6.

Example 4 Detection of Bacillus subtilis from LB Growth Medium

Bacillus subtilis cells grown overnight in 2.5 ml of LB-medium (10 gtryptone, 5 g yeast extract and 10 g NaCl per liter, pH 7.0) wereserially diluted to LB-medium. One ml of each dilution was filteredthrough a 0.22 μm pore size filter. The cells on filter were washed with1 ml of sterile water, the flow direction through the filter wasreversed and the cells were flushed with 0.5 ml of sterile water. 5 μlof the eluate was used as template in PCR reactions that had thefollowing conditions: 1.5 U AmpliTaq Gold DNA Polymerase, 1×PCR bufferII and 6.5 mM MgCl2 (Applied Biosystems, US), 0.8 mM dNTPs (AmershamBiosciences), 0.5 μM Bacillus primers, 1.7 μM Bacillus probe and 41.5 μMBacillus quencher in a total volume of 50 μl. The thermal cyclingprofile was 95° C. 10 min, 95° C. 15 s, 53° C. 30 s and 61° C. 30 srepeated for a total of 40 cycles. In the end of each of the last 20cycles the temperature was briefly lowered to 35° C. for time-resolvedfluorescence measuring. The amount of Bacillus cells in each of theserial dilutions was determined with platings. PCR results were plottedagainst plating results in order to obtain the standard curve presentedin FIG. 7.

Example 5 Detection of Bacillus Subtilis Endospores from Potato Flour

Dilutions of Bacillus subtilis spores were made to Ringer solution (8.6g NaCl, 0.3 g KCl, 0.48 g CaCl2 per liter) containing 10% potato floursuspension. The dilutions were first prefiltered through 5 μm pore sizefilters and then the cells were collected by passing the samples through0.45 μm pore size filters. The spores on filter were washed with 1 ml ofsterile water, the flow direction through the filter was reversed andthe spores were flushed with 1 ml of sterile water. 5 μl of the eluatewas added to PCR reactions as template. The PCR analysis was done as inexample 4. The amount of Bacillus spores in each of the samples wasdetermined with platings. PCR results were plotted against platingresults in order to obtain the standard curve presented in FIG. 8.

Example 6 Extraction of Leucocytes from Whole Blood

An aliquot of 300 μl of whole EDTA blood was mixed with 900 μl of 20 mMTris-HCl, pH 7.5 in order to lyse the red blood cells. Leucocytes werethen collected by filtration through a 5 μm pore size filter. Theleucocytes on the filter were washed with 3 ml of the above buffer, theflow direction through the filter was reversed and the cells flushedfrom the filter with 1 ml of sterile water. Aliquots from 1 to 10 μlwere used as templates in 50 μl PCR reactions that contained 2.0 UAmpliTaq Gold DNA Polymerase, 1×PCR buffer II, 3.5 mM MgCl₂, 0.2 mMdNTPs and 0.5 μM Actine primers. The thermal cycling profile was 95° C.10 min, 95° C. 30 s, 60° C. 30 s and 72° C. 30 s repeated for a total of40 cycles. The PCR reactions were analyzed with agarose gelelectrophoresis. The picture of the gel is shown in FIG. 9. The resultsshow that the 136 bp actin fragment gets amplified only in the presenceof the extracted leucocytes.

It will be appreciated that the methods of the present invention can beincorporated in the form of a variety of embodiments, only a few ofwhich are disclosed herein. It will be apparent for the specialist inthe field that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

1. A nucleic acid amplification assay for quantitative and/orqualitative analysis of the presence of a specific analyte or specificanalytes in a biological sample, which analytes, if present, arecontained in biological particles (4) of said sample (2), in which assaythe sample (2) is forced in a first direction through a filter (6) thatretains said biological particles (4) characterised in that saidbiological particles (4) retained in said filter (6) are flushed, by aflush flow (8), in a second opposite direction through said filter (6)out of said filter (6) and said flush flow (8) containing saidbiological particles (4) flushed out is analysed for the analyte oranalytes.
 2. The assay of claim 1 characterised in that said assaycomprises an additional filtration prior to the filtration retaining thebiological particles (4) containing the analyte or analytes, whichadditional filtration does not retain the biological particles (4)containing the analyte or analytes but retains particles (10) that mightinterfere with the analysis of the analyte or analytes.
 3. The assay ofclaim 1 or 2 characterised in that the flow containing the biologicalparticles (4) containing the analyte or analytes flushed out is analysedfor the analyte or analytes without any further purification.
 4. Theassay of claim 1, 2 or 3 characterised in that retention of thebiological particles (4) containing the analyte or analytes in thefilter (6) is essentially size dependent.
 5. The assay of any of claims1 to 4 characterised in that retention of the biological particles (4)containing the analyte or analytes in the filter (6) is essentiallydependent on the chemical properties of the particle.
 6. The assay ofany of claims 1 to 5 characterised in that the biological particles (4)containing the analyte or analytes are selected from the groupconsisting of prokaryotic or eukaryotic cells or spores or componentsthereof, viruses or viral particles, complexes comprising protein and/ornucleic acid, and any combination thereof.
 7. The assay of claim 6characterised in that the biological particles (4) containing theanalyte or analytes are selected from the group consisting of bacteria,bacterial cell, plant pollen, mithochondria, chloroplast, cell nuclei,virus, phage, chromosome and ribosome.
 8. The assay of any of claims 1to 7 characterised in that the means of analysing the analyte oranalytes is selected from the group consisting of polymerase chainreaction (PCR), reverse transcriptase polymerase chain reaction(RT-PCR), ligase chain reaction (LCR), proximity ligation assay, nucleicacid sequence based amplification (NASBA), strand displacementamplification (SDA) and any combination thereof.
 9. The assay of any ofclaims 1 to 8 characterised in that the biological particles (4)containing the analyte or analytes are flushed with a liquid or a gaspreferably not contained in the original sample
 2. 10. The assay of anyof claims 1 to 9 characterised in that the analyte or analytes areselected from the group consisting of a living and/or dead cell orvirus; a peptide, a protein or complex thereof; a nucleic acid; and anycombination thereof.
 11. The assay of claim 10 characterised in that theanalyte or analytes comprises living and/or dead cells and/or virusesselected from the group consisting of a mold, a yeast, a eukaryotic cellor organism, a pathogenic virus and a cancer cell.
 12. The assay ofclaim 10 characterised in that the analyte or analytes comprises nucleicacids selected from the group consisting of DNA, RNA and any derivativethereof.
 13. The assay of claim 10 characterised in that the analyte oranalytes comprises peptides and/or proteins or complexes thereofselected from the group consisting of a hormone, a growth factor, anenzyme or parts thereof and/or complexes thereof; and any combinationthereof.
 14. An arrangement (12) for preparing a biological sample (2)for quantitative and/or qualitative analysis of the presence of aspecific analyte or specific analytes, which analytes, if present, arecontained in biological particles (4) of the sample (2), wherein thearrangement (12) comprises a) a housing (14) for a filter (6); b) afilter (6) within said housing (14) for retaining the biologicalparticles (4) containing the analyte or analytes, said filter (6) havingtwo sides, i) a sample inlet side (16) and ii) a flushing flow inletside (18); and c) means for i) leading (20) the sample (2) through thefilter (6) from the sample inlet side (16) to the flushing flow inletside (18), ii) leading (22) the flush flow (8) from its inlet side (18)to the sample inlet side (16), and iii) retrieving (24) for analysisbiological particles (4) containing the analyte flushed from the filter(6); characterised in that the arrangement (12) comprises a filter rack(32) that is a multi-way valve, with connections for sample inlet (20),sample retrieval (24), flush flow inlet (36) and waste disposal (38),and optionally for wash flow (34), and the filter rack (32) with thefilter (6) can be turned in alternative positions so that flow isdirected from d) the sample inlet (20) into the filter (6) from thesample inlet side (16) to the flush flow inlet side (18) and to waste(38) or optionally for use as flush flow, e) the flush flow inlet (22)into the filter (6) from the flush flow inlet side (18) to the sampleinlet side (16) and to sample retrieval (24), or f) optionally, the flowinlet (30) into the filter (6) from the sample inlet side (16) to theflush flow inlet side (18) and to waste (38) or for recycling.
 15. Thearrangement (12) according to claim 14 characterised in that thearrangement (12) further comprises a) an additional filter (26) thatdoes not retain the biological particles (4) containing the analyte oranalytes but retains particles (10) that might interfere with theanalysis of the analyte or analytes, and b) means for leading (28) thesample (2) through said additional filter (26) prior to leading itthrough the filter (6) for retaining the biological particles (4)containing the analyte or analytes.
 16. The arrangement (12) accordingto claim 14 or 15 characterised in that the arrangement (12) furthercomprises means for leading (30) a washing liquid or gas through thefilter (6) from the sample inlet side (16) to the flushing flow inletside (18) for washing the retained biological particles (4) containingthe analyte or analytes prior to flushing them out of the filter (6).17. A kit of parts, components and/or reagents for performing the assayaccording to any of claims 1 to
 13. 18. A kit of parts according toclaim 17, characterised in that it comprises the arrangement (12)according to any of claims 14 to 16.